Phenolic polymers like polyphenols and polyphenylenes have several industrial applications including electrical insulation, specialty membranes, and packings but are typically synthesized under harsh reaction conditions and require hazardous chemicals like formaldehyde. Hydroxycinnamic acids, such as p-coumaric acid (p-CA), are aromatic derivatives of lignin hydrolysates, an underutilized and promising renewable feedstock for production of phenolics and phenolic polymers. Recently a strain of Corynebacterium glutamicum has been created by the Joint BioEnergy Institute (JBEI) which expresses phenolic acid decarboxylase (PAD), an enzyme which catalyzes the reaction of p-CA to 4-vinylphenol (4-VP). Further, a deletion of the phdA gene prevents assimilation of p-CA, thereby increasing 4-VP yield. 4-VP is a substituted phenol which can be polymerized to poly(4-vinylphenol) (PVP) in the presence of ligninolytic enzymes like laccases or peroxidases. This work explores in situ polymerization of 4-VP to PVP by supplementing ligninolytic enzymes during fermentation. Cultured in the presence of p-CA, the engineered C. glutamicum strain achieved a maximum 4-VP yield of 45.2%, 57.9%, and 34.7% when fed 2, 5, and 10 g/L p-CA, respectively. Low yield can be attributed to photodegradation of 4-VP and accumulation of the native laccase present in C. glutamicum which may form only dimers and trimers. To further investigate carbon utilization in the cell, the engineered strain was plasmid cured thus removing the PAD enzyme and fermentations for 13C pathway analysis was performed. Polymerization experiments were performed and the polymer was characterized using GPC.

Metabolic engineering has emerged as a highly effective approach to optimizing industrial fermentation processes by introducing purposeful genetic alterations using recombinant DNA technology. Successful metabolic engineering begins with a careful investigation of cellular function, and based on the outcomes of this analysis, an improved strain is created and then constructed using genetic engineering. By modifying the genetic makeup of cells, can increase the production of important chemicals, biofuels, medications, and agricultural products. The most often used genetic engineering tool is plasmid-based gene editing. In plasmid-based gene editing, the desired gene sequence is flanked by similar genome sequences, which encourages the foreign gene's integration into the genome. The main flaw of plasmid-based editing is the presence of selectable markers in the integrated DNA, which impacts cell stability as well as downstream applications that are critical to industries. Recently, with the growth of science, the new gene-editing technology CRISPR (clustered regularly interspaced short palindromic repeat) has revolutionized the field of gene editing. It has been used to incorporate the foreign genes into the genome of the microbial host without any mark and has more efficiency than the plasmid-based gene editing technique. CRISPR is utilized to achieve markerless integration of genes in genomes of microbes, which promotes cell stability and is also especially beneficial for applications in industries. In this experiment successfully integrated two genes into the genome of C.glutamicum employing markerless integration via homologous recombination, allowing cells to metabolize acetate into acetyl-CoA and improve the conversion of pyruvate into lactate. Further, this strain of C.glutamicum can be utilized as a platform for producing ethyl lactate, a green solvent using a microbial host

Lignocellulose, the major structural component of plant biomass, represents arenewable substrate of enormous biotechnological value. Microbial production of chemicals from lignocellulosic biomass is an attractive alternative to chemical synthesis. However, to create industrially competitive strains to efficiently convert lignocellulose to high-value chemicals, current challenges must be addressed. Redox constraints, allosteric regulation, and transport-related limitations are important bottlenecks limiting the commercial production of renewable chemicals from lignocellulose. Advances in metabolic engineering techniques have enabled researchers to engineer microbial strains that overcome some of these challenges but new approaches that facilitate the commercial viability of lignocellulose valorization are needed. Biological systems are complex with a plethora of regulatory systems that must be carefully modulated to efficiently produce and excrete the desired metabolites. In this work, I explore metabolic engineering strategies to address some of the biological constraints limiting bioproduction such as redox, allosteric, and transport constraints to facilitate cost-effective lignocellulose bioconversion.

Alkanolamines are useful as building blocks for a variety of applications, ranging from medical applications such as drug and gene delivery. In this work, Escherichia coli was investigated as a viable candidate for the production of 5-amino-1-pentanol (5-AP). Taking advantage of the existing L-lysine degradation pathway, a novel route to 5-AP was constructed by co-expressing the genes cadA (encoding lysine decarboxylase, responsible for the conversion of L-lysine to cadaverine) and patA (encoding putrescine aminotransferase, responsible for the conversion of cadaverine to 5-amino-1-pentanal), followed by the endogenous reduction of 5-amino-pentanal (5-APL) to 5-AP. To avoid the competing conversion of 5-APL to 5-amino-1-pentanoate and avoid accumulation of byproduct 1-Δ-piperideine, further host engineering was performed to delete the gene patD also known as prr (encoding 5-amino-pentanal dehydrogenase). Flask scale fermentation experiments in minimal medium of the newly constructed pathway was conducted where 62.6 mg/L 5-AP was observed to be produced. It was hypothesized that 5-AP production could be boosted by optimizing production medium to M10 media. However, change in the culture medium resulted in the production of just 51 mg/L 5-AP. Shifts observed in HPLC chromatogram peaks made it difficult to conclude exact titers of 5-AP and can be further improved by exploring different analysis methods and optimization of the method currently in place.

Lignin is a naturally abundant source of aromatic carbon but is largely underutilized inindustry because it is difficult to decompose. Recent research activity has targeted the development of a biological platform for the conversion of lignin and lignin-derived feedstock. Corynebacterium glutamicum is a standout candidate for the bacterial depolymerization and assimilation of lignin because of its performance as an industrial producer of amino acids, resistance to aromatic compounds in lignin, and low extracellular protease activity. Under the current study, nine experimental strains of C. glutamicum were engineered with sequencing-confirmed plasmids to overexpress and secrete lignin-modifying enzymes with the eventual goal of using lignin as raw feed for the sustainable production of valuable chemicals. Within the study, laccase and peroxidase activity were discovered to be decreased in C. glutamicum culture media. For laccase the decrease reached statistical significance, with an activity of about 10.9 U/L observed in water but only about 7.56 U/L and 7.42 U/L in fresh and spent BHI media, respectively, despite the same amounts of enzyme being added. Hypothesized reasons for this inhibitory effect are discussed here, but further work is needed to identify causative factors and realize the potential of C. glutamicum for waste biomass valorization.

Amino acids and related targets are typically produced by well-characterized heterotrophs including Corynebacterium glutamicum and Escherichia coli. Recent efforts have sought to supplant these sugar-intensive processes through the metabolic engineering of cyanobacteria, which instead can directly utilize atmospheric carbon dioxide (CO2) and sunlight. One of the most promising among recently discovered photoautotrophic strains is Synechococcus elongatus UTEX 2973 (hereafter UTEX 2973), which has been reported to have doubling times as low as 1.5 hours. While encouraging, there are still major challenges preventing the widespread industrial acceptance of engineered cyanobacteria, chief among them is the scarcity of genetic tools and parts with which to engineer production strains. Here, UTEX 2973 was engineered to overproduce L-lysine through the heterologous expression of feedback-resistant copies of aspartokinase lysC and the L-lysine exporter ybjE from Escherichia coli, as aided by the characterization of novel combinations of genetic parts and expression sites. At maximum, using a plasmid-based expression system, a L-lysine titer of 556 ± 62.3 mg/L was attained after 120 hours, surpassing a prior report of photoautotrophic L-lysine bioproduction. Modular extension of the pathway then led to the novel photosynthetic production of the corresponding diamine cadaverine (55.3 ± 6.7 mg/L by 96 hours) and dicarboxylate glutarate (67.5 ± 2.2 mg/L by 96 hours). Lastly, mass transfer experiments were carried out to determine if the solubility of CO2 in and its rate of mass transfer to BG-11 media could be improved by supplementing it with various amines, including cadaverine. Ultimately, however, cyanobacteria grown in the presence of all tested amines was worse than in BG-11 alone, demonstrating the need for additional tolerance engineering to successfully implement this strategy.

The objective of this research was to develop Aluminophosphate-five (AlPO4-5, AFI) zeolite adsorbents for efficient oxygen removal from a process stream to support an on-going Department of Energy (DOE) project on solar energy storage. A molecular simulation study predicted that substituted AlPO4-5 zeolite can adsorb O2 through a weak chemical bond at ambient temperature. Substituted AlPO4-5 zeolite was successfully synthesized via hydrothermal crystallization by following carefully designed procedures to tailor the zeolite for efficient O2 adsorption. Synthesized AlPO4-5 in this work included Sn/AlPO-5, Mo/AlPO-5, Pd/AlPO-5, Si/AlPO-5, Mn/AlPO-5, Ce/AlPO-5, Fe/AlPO-5, CuCe/AlPO-5, and MnSnSi/AlPO-5. While not all zeolite samples synthesized were fully characterized, selected zeolite samples were characterized by powder x-ray diffraction (XRD) for crystal structure confirmation and phase identification, and nitrogen adsorption for their pore textural properties. The Brunauer-Emmett-Teller (BET) specific surface area and pore size distribution were between 172 m2 /g - 306 m2 /g and 6Å - 9Å, respectively, for most of the zeolites synthesized. Samples of great interest to this project such as Sn/AlPO-5, Mo/AlPO-5 and MnSnSi/AlPO-5 were also characterized using x-ray photoelectron spectroscopy (XPS) and energy-dispersive x-ray spectroscopy (EDS) for elemental analysis, scanning electron microscopy (SEM) for morphology and particle size estimation, and electron paramagnetic resonance (EPR) for nature of adsorbed oxygen. Oxygen and nitrogen adsorption experiments were carried out in a 3-Flex adsorption apparatus (Micrometrics) at various temperatures (primarily at 25℃) to determine the adsorption properties of these zeolite samples as potential adsorbents for oxygen/nitrogen separation. Experiments showed that some of the zeolite samples adsorb little-to-no oxygen and nitrogen at 25℃, while other zeolites such as Sn/AlPO-5, Mo/AlPO-5, and MnSnSi/AlPO-5 adsorb decent but inconsistent amounts of oxygen with the highest observed values of about 0.47 mmol/ g, 0.56 mmol/g, and 0.84 mmol/ g respectively. The inconsistency in adsorption is currently attributed to non-uniform doping of the zeolites, and these findings validate that some substituted AlPO4-5 zeolites are promising adsorbents. However, more investigations are needed to verify the causes of this inconsistency to develop a successful AlPO4-5 zeolite-based adsorbent for oxygen/nitrogen separation.

Bioconversion of lignocellulosic sugars is often suboptimal due to global regulatory mechanisms such as carbon catabolite repression and incomplete/inefficient metabolic pathways. While conventional bioprocessing strategies for metabolic engineering have predominantly focused on a single engineered strain, the alternative development of synthetic microbial communities facilitates the execution of complex metabolic tasks by exploiting unique community features (i.e., modularity, division of labor, and facile tunability). In this dissertation, these features are leveraged to develop a suite of generalizable strategies and transformative technologies for engineering Escherichia coli coculture systems to more efficiently utilize lignocellulosic sugar mixtures. This was achieved by rationally pairing and systematically engineering catabolically-orthogonal Escherichia coli sugar specialists. Coculture systems were systematically engineered, as derived from either wild-type Escherichia coli W, ethanologenic LY180, lactogenic TG114 or succinogenic KJ122. Net catabolic activities were then readily balanced by simple tuning of the inoculum ratio between sugar specialists, ultimately enabling improved co-utilization (98% of 100 g L-1 total sugars) of glucose-xylose mixtures (2:1 by mass) under simple batch fermentation conditions. We next extended this strategy to a coculture-coproduction system capable of capturing and fixing CO2 evolved during biofuel production through inter-strain metabolic cooperation. Holistically, this work contributes to an improved understanding of the dynamic behavior of synthetic microbial consortia as enhanced bioproduction platforms and carbon conservation strategy for renewable fuels and chemicals from non-food carbohydrates

Metabolic engineering of bacteria has become a viable technique as a sustainable and efficient method for the production of biochemicals. Two main goals were explored: investigating styrene tolerance genes in E. coli and engineering cyanobacteria for the high yield production of L-serine. In the first study, genes that were shown to be highly differentially expressed in E. coli upon styrene exposure were further investigated by testing the effects of their deletion and overexpression on styrene tolerance and growth. It was found that plsX, a gene responsible for the phospholipid formation in membranes, had the most promising results when overexpressed at 10 µM IPTG, with a relative OD600 of 706 ± 117% at 175 mg/L styrene when compared to the control plasmid at the same concentration. This gene is likely to be effective target when engineering styrene- and other aromatic-producing strains, increasing titers by reducing their cytotoxicity.In the second study, the goal is to engineer the cyanobacterium Synechococcus sp. PCC 7002 for the overproduction of L-serine. As a robust, photosynthetic bacteria, it has potential for being used in such-rich states to capture CO2 and produce industrially relevant products. In order to increase L-serine titers, a key degradation gene, ilvA, must be removed. While ilvA is responsible for degrading L-serine into pyruvate, it is also responsible for initiating the only known pathway for the production of isoleucine. Herein, we constructed a plasmid containing the native A0730 gene in order to investigate its potential to restore isoleucine production. If functional, a Synechococcus sp. PCC 7002 ΔilvA strain can then be engineered with minimal effects on growth and an expected increase in L-serine accumulation.

In the past, the photovoltaic (PV) modules were typically constructed with glass superstrate containing cerium oxide and EVA (ethylene vinyl acetate) encapsulant containing UV absorbing additives. However, in the current industry, the PV modules are generally constructed without cerium oxide in the glass and UV absorbing additives in EVA to increase quantum efficiency of crystalline silicon solar cells in the UV regions. This new approach is expected to boost the initial power output of the modules and reduce the long-term encapsulant browning issues. However, this new approach could lead to other durability and reliability issues such as delamination of encapsulant by damaging interfacial bonds, destruction of antireflection coating on solar cells and even breakage of polymeric backbone of EVA. This work compares the durability and reliability issues of PV modules having glass without cerium oxide and EVA with (aka, UVcut or UVC) and without (aka, UVpass or UVP) UV absorbing additives. In addition, modules with UVP front and UVC back EVA have also been investigated (aka, UVhybrid or UVH). The mini-modules with nine split cells used in this work were fabricated at ASU’s Photovoltaic Reliability Laboratory. The durability and reliability caused by three stress variables have been investigated and the three variables are temperature, humidity/oxygen and UV dosage. The influence of up to 800 kWh/m2 UV dosage has been investigated at various dosage levels. Many material and device characterizations have been performed to ascertain the degradation modes and effects. The UVC modules showed encapsulant discoloration at the cell centers as expected but the UVH modules showed a ring-shaped encapsulant discoloration close to the cell edges as evidenced in the UV fluorescence (UVF) imaging study. The PV modules containing UVP on both sides of cells with limited access to humidity or oxygen through backsheet (covered backsheet with adhesive aluminum tape) seem to experience encapsulant delamination as evidenced in the UVF images. Plausible explanations for these observations have been presented.